Interaction of H+ and Zn2+ on recombinant and native rat neuronal GABAA receptors

Abstract

1. The interaction of Zn2+ and H+ ions with GABAA receptors was examined using Xenopus laevis oocytes expressing recombinant GABAA receptors composed of subunits selected from alpha1, beta1, gamma2S and delta types, and by using cultured rat cerebellar granule neurones. 2. The potency of Zn2+ as a non-competitive antagonist of GABA-activated responses on alpha1beta1 receptors was reduced by lowering the external pH from 7.4 to 5.4, increasing the Zn2+ IC50 value from 1.2 to 58.3 microM. Zinc-induced inhibition was largely unaffected by alkaline pH up to pH 9.4. 3. For alpha1beta1delta subunits, concentration-response curves for GABA were displaced laterally by Zn2+ in accordance with a novel mixed/competitive-type inhibition. The Zn2+ IC50 at pH 7.4 was 16.3 microM. Acidification of Ringer solution resulted in a reduced antagonism by Zn2+ (IC50, 49.0 microM) without affecting the type of inhibition. At pH 9.4, Zn2+ inhibition remained unaffected. 4. The addition of the gamma2S subunit to the alpha1beta1delta construct caused a marked reduction in the potency of Zn2+ (IC50, 615 microM), comparable to that observed with alpha1beta1gamma2S receptors (IC50 639 microM). GABA concentration-response curves were depressed in a mixed/non-competitive fashion. 5. In cultured cerebellar granule neurones, Zn2+ inhibited responses to GABA in a concentration-dependent manner. Lowering external pH from 7.4 to 6.4 increased the IC50 from 139 to 253 microM. 6. The type of inhibition exhibited by Zn2+ on cerebellar granule neurones, previously grown in high K+-containing culture media, was complex, with the GABA concentration-response curves shifting laterally with reduced slopes and similar maxima. The Zn2+-induced shift in the GABA EC50 values was reduced by lowering the external pH from 7.4 to 6.4. 7. The interaction of H+ and Zn2+ ions on GABAA receptors suggests that they share either a common regulatory pathway or coincident binding sites on the receptor protein. The apparent competitive mode of block induced by Zn2+ on alpha1beta1delta receptors is shared by GABAA receptors on cerebellar granule neurones, which are known to express delta-subunit-containing receptors. This novel mechanism is masked when a gamma2 subunit is incorporated into the receptor complex, revealing further diversity in the response of native GABAA receptors to endogenous cations.

Figure 1. Sensitivity of the Zn²⁺ inhibition of GABA-activated conductances in Xenopus oocytes expressing α1β1 GABA_A receptors

Membrane currents and conductance changes evoked by bath-applied 10 μm GABA in the absence and presence of 1 μm zinc at pH 7.4 (A), 5.4 (B) and 9.4 (C), respectively. The GABA-activated responses in all oocytes increased at low pH and decreased at high pH. Records are selected from two oocytes. Current calibration of 100 nA corresponds to A and B, and 250 nA corresponds to C. GABA was applied for the duration indicated by the horizontal bars. Recovery responses were obtained after 3 min in Ringer solution at the respective external pH values following exposure to Zn²⁺. Membrane conductance was ascertained by regularly applying hyperpolarizing voltage-clamp commands (−10 mV amplitude, 1 s duration, frequency 0.2 Hz) from the holding potential of −40 mV.

Figure 2. Concentration-response relationships for Zn²⁺ inhibition of GABA-activated conductances on α1β1 GABA_A receptors

A, left panel, normalized zinc concentration-inhibition curves were constructed for GABA at external pH 5.4, 7.4 and 9.4. For this and all subsequent zinc inhibition curves, the GABA-induced conductances were normalized to the response evoked by 10 μm GABA in the absence of zinc. All data were fitted according to the antagonist inhibition model (see Methods) and IC₅₀ values determined (Table 1; n = 9). The points represent the means ± s.e.m. A, right panel and B, indicate GABA equilibrium concentration conductance curves constructed in the absence and presence of 1 μm zinc at pH 7.4 and 9.4, or 50 μm zinc at pH 5.4 and normalized to the response induced by 10 μm GABA in the absence of zinc at pH 7.4. The data were fitted with the logistic equation (see Methods; n = 18) and EC₅₀ values and Hill coefficients determined (Tables 2 and 3).

Figure 3. Modulation by external pH of Zn²⁺ inhibition of GABA-induced responses recorded from oocytes expressing GABA_A receptor α1β1δ subunits

A, left panel, zinc concentration-inhibition relationships constructed at external pH 5.4, 7.4 and 9.4, and fitted according to the antagonist inhibition model and IC₅₀ values determined (Table 1; n = 9). A, right panel, GABA equilibrium concentration-response curves constructed in the absence and presence of 10 and 100 μm Zn²⁺ at pH 7.4. B, GABA equilibrium concentration-response curves constructed in the absence and presence of 50 μm zinc (left, pH 5.4) and 10 μm zinc (right, pH 9.4). The EC₅₀ values and Hill coefficients for GABA were determined from the logistic model (Tables 2 and 3; n = 20).

Figure 4. Regulation of α1β1γ2S and α1β1γ2Sδ GABA_A receptor function by Zn²⁺

A and B, left panels, zinc concentration-inhibition relationships constructed for 10 μm GABA-activated responses recorded from α1β1γ2S and α1β1γ2Sδ GABA_A receptors, respectively. All data were fitted according to the antagonist inhibition model and IC₅₀ values determined (Table 1, n = 6). A and B, right panels, GABA equilibrium concentration-response curves in the absence and presence of 5 and 300 μm or 10 and 300 μm zinc recorded from oocytes expressing α1β1γ2S and α1β1γ2Sδ GABA_A receptors, respectively. The EC₅₀ values and Hill coefficients for GABA were determined from the logistic model (Tables 2 and 3, n = 12).

Figure 5. Modulation of GABA-activated currents in cultured cerebellar granule neurones by H⁺ and Zn²⁺ ions

A, whole-cell membrane currents activated by 10 μm GABA before and after the application of 10–500 μm Zn²⁺ at external pH values of 7.4 (upper panel) and 6.4 (lower panel). Recovery responses were obtained in control Krebs solution 5 min after Zn²⁺ application at the respective external pH. Records are from two neurones. The 100 pA calibration bar applies to the upper records. The holding potential was −40 mV. B, zinc concentration-inhibition curves for the antagonism of the 10 μm GABA-activated response at external pH values of 7.4 (^) or 6.4 (•) constructed from fifteen cells. The curves were generated from the inhibition model (see Methods), but since the inhibition saturated at approximately 20% of the control GABA-activated response, a constant was incorporated and the IC₅₀ values were determined.

Figure 6. Inhibition by Zn²⁺ of GABA-activated responses at pH 7.4 on cerebellar granule neurones

A, whole-cell currents recorded at −40 mV holding potential for different concentrations of GABA (1 μm to 2.5 mm) in the absence (upper panel) and presence of 300 μm Zn²⁺ (lower panel). B, analysis of GABA concentration-response curves at pH 7.4 in the absence (^) and presence (•) of 300 μm Zn²⁺. The continuous line curve fit was generated by the agonist logistic model (see Methods). The dotted lines illustrate the corresponding curves for data obtained at pH 6.4 (see Fig. 7).

Figure 7. GABA-activated responses at pH 6.4 on cerebellar granule neurones: inhibition by Zn²⁺

A, whole-cell currents recorded for 1 μm to 2.5 mm GABA in the absence (upper panel) and presence of 300 μm Zn²⁺ (lower panel). Holding potential, −40 mV. B, GABA concentration-response curves are plotted at pH 6.4 in the absence (^) and presence (•) of 300 μm Zn²⁺. The continuous line curve fit was provided by the logistic model (see Methods). For comparison, the dotted lines plot the corresponding curves for data obtained at pH 7.4 (see Fig. 6).